The Potential of Fecal and Urinary Biomarkers for Early Detection of Pancreatic Ductal Adenocarcinoma: A Systematic Review

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer often diagnosed at advanced stages, highlighting the urgent need for early detection strategies. This systematic review explores the potential of fecal and urinary biomarkers for early PDAC detection. A comprehensive search identified eight relevant studies investigating various biomarkers, including proteins, metabolites, microbial profiles, DNA mutations, and non-coding RNAs. Promising findings suggest that urinary biomarkers related to metabolic alterations, inflammatory processes, fecal microbiome profiles, and fecal miRNAs hold diagnostic potential even at early stages of PDAC. Combining biomarkers into panels may enhance diagnostic accuracy. Challenges such as validation in larger cohorts, standardization of protocols, and regulatory approval must be addressed for clinical translation. Despite these hurdles, non-invasive urinary and fecal biomarkers represent a promising avenue for improving PDAC outcomes through early detection.


Introduction And Background
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers, with a five-year survival rate of only 9% [1].Most patients are diagnosed at an advanced stage when curative surgery is no longer possible, contributing to the dismal prognosis [2].Early detection of PDAC is critical to improve patient outcomes, as surgical resection at an early stage offers the only potential cure [3].In recent years, research has focused on identifying biomarkers in biofluids other than blood, such as urine and stool, which may detect molecular changes associated with early carcinogenesis.These non-invasive tests have the advantages of being costeffective, reproducible, and convenient for patients [4].Several potential fecal and urinary biomarkers for early PDAC detection have been identified, including proteins, metabolites, DNA mutations, and microRNAs (miRNAs) [5].
Urinary biomarkers are attractive candidates as urine collection is simple, and urinary molecular changes may reflect systemic alterations during PDAC development [6].Urinary metabolites related to glucose metabolism, inflammation, and cachexia have shown potential for early PDAC detection [7].Urinary exosomal miRNAs, stable, extracellular miRNAs encapsulated in vesicle nanoparticles, have also shown diagnostic potential [8].
Likewise, fecal biomarkers are promising as molecular alterations in pancreatic secretions may be detectable in stool long before symptoms arise [9].Fecal microbial profiles are altered in PDAC patients, and PDAC can be distinguished from healthy controls [10].Specific bacterial species and fecal volatile organic compounds may serve as early diagnostic indicators of PDAC [11].Additionally, differential expression of fecal miRNAs has been detected in PDAC patients compared to healthy individuals [12].
In summary, evidence suggests that fecal and urinary biomarkers could enable earlier PDAC detection than currently possible.Non-invasive urine and stool tests present a novel screening and early diagnosis approach.Further, prospective studies are warranted to validate the clinical utility of these biomarkers.Systematic reviews summarizing the current evidence on these emerging biomarkers are important to guide future research and facilitate potential translation into clinical practice for earlier PDAC detection and improved patient outcomes.

Search Strategy
Our search strategy was developed in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines to ensure the thorough identification of studies pertinent to fecal and urinary biomarkers for early detection of pancreatic ductal adenocarcinoma (PDAC).Extensive searches were performed across several electronic databases, including PubMed, Medline, Embase, and the Cochrane Library, with the timeframe extending from the inception of each database to December 2023.To ensure the inclusion of grey literature and ongoing research, our strategy extended to scrutinizing the reference lists of selected articles and exploring clinical trial registries and relevant conference proceedings.The comprehensiveness of our search was validated by a medical information retrieval specialist, confirming the alignment of our methodology with the PRISMA guidelines.

Eligibility Criteria
The eligibility criteria for our systematic review were meticulously defined to ensure the selection of relevant and high-quality studies.Our inclusion criteria are tailored to peer-reviewed research articles, casecontrol studies, cohort studies, and clinical trials that examine fecal and urinary biomarkers in early pancreatic ductal adenocarcinoma (PDAC) detection.We consider studies that evaluate the diagnostic performance of these biomarkers, including metrics such as accuracy, sensitivity, and specificity, and those that focus on developing diagnostic algorithms or models.
Our review encompasses studies focusing on three main pancreatic ductal adenocarcinoma (PDAC) objectives.Firstly, we examine research exploring the correlation between fecal or urinary biomarkers and early-stage PDAC to elucidate potential diagnostic indicators.Secondly, we evaluate the feasibility of implementing these biomarkers in clinical environments for the purpose of screening individuals for PDAC, thereby potentially enhancing early detection rates.Lastly, we prioritize studies that rigorously validate these biomarkers' efficacy by ensuring adequate control groups' presence, thereby enhancing the reliability and robustness of the findings.Through this comprehensive analysis, we aim to contribute insights that could inform advancements in PDAC detection and management strategies.Only studies published in English were included to ensure consistency in the review process.
Conversely, our exclusion criteria remove articles that do not directly assess fecal or urinary biomarkers for PDAC.Studies focusing solely on other forms of cancer or non-cancerous pancreatic diseases were excluded to maintain a clear focus on PDAC.Similarly, articles based on animal models, case reports, editorials, commentary, and grey literature, including conference abstracts, posters, and unpublished works, were also excluded.This decision ensured that the data synthesized in our review came from studies with the most robust methodological designs relevant to human clinical outcomes in the context of early PDAC detection.
By adhering to these eligibility criteria, our review ensures the inclusion of studies that are most pertinent to advancing the understanding and clinical application of fecal and urinary biomarkers in the early detection of PDAC.

Data Extraction
A systematic data extraction methodology has been implemented to guarantee accuracy and thoroughness in our systematic review.The initial phase involves an assessment of articles based on titles and abstracts.This task is executed by two independent reviewers who categorize each article as "relevant", "probably relevant", or "not relevant".Such a preliminary filtering stage is fundamental to identifying articles that warrant more detailed examination.
Following this, full-text articles classified as "relevant" or "probably relevant" are retrieved for comprehensive evaluation.Data from these articles is extracted using a standardized data collection template created in Microsoft Excel (Microsoft, Redmond, Washington).This approach promotes the consistency of the data extraction across all studies.
In parallel, the independent reviewers utilize the predefined inclusion and exclusion criteria to assess each article's eligibility.When discrepancies between the two reviewers arise, they are resolved by consulting a third independent reviewer, ensuring a consensus is reached and thus maintaining the integrity of the data extraction process.
The data extraction template is meticulously designed to capture essential information pertinent to our research question.It includes fields for author(s), year of publication, biomarker(s) studied, study design, population size, and key metrics of diagnostic performance such as accuracy, sensitivity, and specificity.Moreover, it collates the primary findings and any recommendations made by the study.This detailed data extraction procedure enables the construction of a comprehensive database, facilitating subsequent analysis and synthesis of the extracted data and ensuring no significant detail is overlooked.
This rigorous data extraction method ensures that the conclusions drawn from our systematic review are based on a complete and accurate representation of the available evidence concerning fecal and urinary biomarkers for early detection of pancreatic ductal adenocarcinoma (PDAC).

Data Analysis and Synthesis
Our review's data analysis and synthesis are structured to methodically evaluate the diagnostic potential of fecal and urinary biomarkers in early pancreatic ductal adenocarcinoma (PDAC) detection.Quantitative data, such as diagnostic performance measures, sensitivity, specificity, and accuracy rates, will be statistically analyzed.Where data homogeneity permits, a meta-analysis will be considered to pool performance metrics and provide a more robust estimation of biomarker efficacy.
Qualitative data encompassing study findings and authors' recommendations will undergo thematic synthesis.This process involves extracting themes related to these biomarkers' utility, applicability, and potential integration into clinical practice.
Integrating quantitative and qualitative analyses will offer a comprehensive view of the current state of fecal and urinary biomarkers in PDAC diagnosis.A narrative synthesis will complement the statistical findings, contextualizing them within the broader research landscape and discussing their implications in line with the existing body of knowledge.
In addition, we will evaluate the evidence's quality and the strength of the synthesized data, identifying any literature gaps or inconsistencies.By adopting this analytical approach, our synthesis aims to provide a concise yet comprehensive understanding of the early detection capabilities of fecal and urinary biomarkers for PDAC, ultimately contributing to advancing non-invasive diagnostic methods in oncology.

Study Selection Process
Our systematic search across multiple electronic databases initially retrieved 121 articles.Upon removing 27 duplicates, we screened 94 records by title and abstract.The screening process was guided by our inclusion criteria, which required studies to focus on fecal and urinary biomarkers to detect PDAC early and to report on diagnostic performance outcomes such as accuracy, sensitivity, and specificity.We excluded studies that were not in English, did not pertain to human clinical outcomes, or focused on other types of cancer or diseases.Studies were also excluded if they were based on animal models, were reviews or editorials, or were grey literature, such as conference abstracts or unpublished studies.
After the screening process, 78 records were excluded, leaving 16 reports that were sought for full-text retrieval.Of these, 13 reports could be retrieved and were subjected to a rigorous assessment for eligibility based on the above criteria, excluding five more studies.
Consequently, eight new studies met our inclusion criteria and were selected for this review.The reference lists of these studies were scrutinized for potentially relevant studies, but this did not yield any additional articles for inclusion.The entire process, from identification to the final inclusion of studies, is depicted in the PRISMA flowchart (Figure 1).This visual aids in transparently illustrating the systematic approach and meticulous criteria we adhered to in selecting studies for our review.

Characteristics of Selected Studies
Our systematic review incorporates eight significant studies that provide valuable information regarding fecal and urinary biomarkers for the early detection of PDAC.The selected studies encompass various research methodologies, including developing diagnostic models, validating biomarker panels, and analyzing biomarker efficacy using different patient cohorts and control groups.
These studies were published between 2014 and 2023 and utilized various analytical techniques, from deep learning models to metagenomic sequencing and mass spectrometry.The patient populations vary in size, from smaller, focused cohorts to larger, more comprehensive collections of samples.The diagnostic performance of the biomarkers studied is quantitatively presented with accuracy, sensitivity, specificity, and other relevant metrics.
A summary of these studies, including the authors, publication year, biomarkers studied, study design, population size, and main findings, is concisely presented in

Discussion
This systematic review has consolidated the evidence on the burgeoning field of fecal and urinary biomarkers for the early detection of pancreatic ductal adenocarcinoma (PDAC), a malignancy notorious for its late presentation and poor prognosis.The discovery of multiple biomarker candidates spanning various biological substrates, such as metabolites, proteins, microbial profiles, DNA mutations, and non-coding RNAs, heralds a new era in the non-invasive detection of this lethal cancer.Historically, the diagnostic journey for PDAC has been fraught with obstacles, mainly due to the absence of specific symptoms and the rapid progression of the disease.Conventional diagnostic tools, such as imaging and tissue biopsies, often only confirm the disease at an advanced stage when therapeutic options are limited and less effective.In contrast, the biomarkers identified in our review could signify a paradigm shift, promising a timely and more precise diagnosis.By potentially detecting the disease when it is still localized within the pancreas, these biomarkers could dramatically improve survival rates by enabling curative surgical interventions and personalized therapeutic strategies.
Urinary biomarkers related to metabolic alterations, inflammation, and cachexia in PDAC demonstrated discriminatory power even at early stages [6,17,18].Certain urinary proteins involved in processes like angiogenesis and extracellular matrix remodeling were specifically upregulated in PDAC patients versus controls [5].Urinary exosomal miRNAs also exhibited diagnostic potential, likely reflecting underlying tumor molecular characteristics [8].Fecal microbiome profiles distinguished PDAC patients from healthy individuals [10].Specific bacterial species and fecal volatile metabolites showed alteration even before symptom onset and could differentiate PDAC.Fecal DNA mutations shed from apoptotic cancer cells into the duodenum offered early diagnostic possibilities [19].Differentially expressed fecal miRNA levels could also detect PDAC earlier than conventional methods [12].
The potential of these non-invasive biomarkers extends beyond the laboratory; they stand as the cornerstone for developing groundbreaking early detection strategies for PDAC, which currently has one of the lowest survival rates among cancers.The urgency of this need cannot be overstated; early-stage detection is directly correlated with increased survival, yet most patients present with advanced disease.The translation of these biomarkers from research to clinical application hinges on robust validation studies that confirm their diagnostic accuracy and evaluate their effectiveness in real-world screening, particularly in asymptomatic individuals who may benefit the most from early detection [14].
The complexity of PDAC pathobiology suggests that a multiplexed approach, leveraging a panel of biomarkers, may capture the heterogeneity of the disease better than any single marker.This synergistic approach could enhance diagnostic accuracy and specificity, thereby minimizing the risk of false positives, which is crucial in cancer screening.However, the successful implementation of such panels necessitates meticulous optimization and standardization of sample collection and processing protocols, which are paramount to ensuring the reproducibility and reliability of results across different populations and clinical settings.Furthermore, analytical platforms used to detect these biomarkers need to be validated for clinical use, a process that must be navigated alongside regulatory bodies to ensure the tests meet the stringent requirements for safety and efficacy before they can be approved for widespread use [15].
Several challenges remain before these biomarkers can be clinically implemented.Larger cohorts across multiple centers are needed to validate preliminary findings and account for potential demographic and geographic variations [20].Standardized sample collection, storage, and analysis protocols must be established to ensure reproducibility.Appropriate cutoffs for distinguishing PDAC must be defined, which may require machine learning approaches applied to big datasets [21].Strategies should be developed to integrate these emerging biomarkers with existing methods, such as cost-effective imaging.
Nonetheless, fecal and urinary biomarkers represent a promising new direction in PDAC early diagnosis.Development of screening approaches combining risk factors like age and smoking with biomarker testing could enable the identification of individuals for targeted investigations, potentially improving detection at resectable stages.Earlier diagnosis would also expand eligibility for emerging therapies.Ultimately, convenient, non-invasive tests using urine or stool could provide an opportunity for routine PDAC screening and detection at curable stages, leading to improved prognosis for this lethal malignancy.It is also important to determine the time during carcinogenesis when these biomarkers become detectable, as extremely early changes may reflect unviable screening targets due to low specificity [22].
Despite the considerable challenges ahead, the advent of non-invasive urinary and fecal biomarkers represents a seismic shift in the landscape of PDAC early detection.These biomarkers have the disruptive potential to revolutionize outcomes by diagnosing PDAC at stages amenable to surgical intervention, the only curative treatment option currently available.With meticulous validation against clinical endpoints and a strategy for seamless integration into existing diagnostic frameworks, these biomarkers could substantially alter the trajectory of this malignancy, shifting it from a near-certain death sentence to a condition with a hopeful prognosis.
The promise these biomarkers hold is not just in their scientific novelty but in their capacity to be integrated into routine screening programs.This could lead to earlier intervention strategies and open new avenues for personalized medicine in PDAC management.As this field matures, it is imperative that the scientific community continues to build upon the groundwork laid out by the studies in this review, overcoming obstacles from bench to clinic to harness the full potential of these biomarkers.
Therefore, the future of PDAC management may lie in the everyday, where indicators of our body's deepest health secrets are captured in what we leave behind.In what may be seen as the most unassuming places, like our toilets and trash cans, lies the key to unlocking a new chapter in the fight against one of the deadliest forms of cancer.The symbol of this future is the non-invasive nature of these diagnostic tools, embodying hope for early detection and a testament to the innovative spirit of medical research.In this regard, the continued investment and focus on the translational research of these biomarkers are not just warranted but imperative.

Conclusions
Fecal and urinary biomarkers offer a non-invasive approach for the early detection of PDAC, addressing critical gaps in current diagnostic strategies.The synthesis of evidence from this systematic review underscores the diagnostic potential of various biomarkers, highlighting the promise of identifying PDAC at earlier, more treatable stages.However, significant challenges remain in terms of validation, standardization, and regulatory approval.Overcoming these hurdles is essential to realize the transformative potential of these biomarkers in clinical practice.As such, rigorous multi-center trials are needed to confirm their efficacy and reliability across diverse populations.Furthermore, integrating these biomarkers into existing screening programs could enhance their practical utility and acceptance in the medical community.
With continued research efforts and strategic implementation, non-invasive urinary and fecal biomarkers could revolutionize PDAC management, ultimately leading to improved patient outcomes and survival rates.
Future research should also focus on the cost-effectiveness of implementing these screening tools in routine healthcare settings, which could be pivotal for their widespread adoption.Additionally, as we advance our understanding of the molecular pathways involved in PDAC, biomarkers could also aid in personalizing treatment strategies, thereby not only detecting the disease earlier but also tailoring interventions to individual patient profiles.The journey from discovery to clinical application is complex and fraught with challenges, but the potential benefits for early detection and management of PDAC could be profound, altering the course of one of the most lethal cancers.

FIGURE 1 :
FIGURE 1: PRISMA flow diagram of selection of studies for inclusion in the systematic review PRISMA -Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA)

Table 1 .
This table is an accessible guide to grasp the diversity and depth of research conducted in this field, providing a solid foundation for a nuanced understanding of the potential of fecal and urinary biomarkers in the early diagnosis of PDAC.

TABLE 1 : A summary of the studies included in this systematic review.
PDAC -pancreatic ductal adenocarcinoma; NAFLD -nonalcoholic fatty liver disease; PC -pancreatic cancer; PCL -pre-cancerous lesion; ELISA -enzyme-linked immunosorbent assay